The overall goal of this project is to understand the mechanisms by which Kir2 inward rectifier potassium channels regulate cardiac excitability. It has been firmly established that members of the Kir2 subfamily underlie cardiac lK1, the major K+ current responsible for stabilizing the resting membrane potential and shaping repolarization of the AP in cardiac myocytes. Mutations in the human Kir2.1 gene were recently found to be the cause of two novel syndromes, Long QT7 and Short QT3. Previous reports and preliminary data also point to lK1 as a major player in the early response of the heart to hypoxia. Nevertheless, the mechanisms of IK1 regulation and the contribution of IK1 to AP under various physiologically important conditions remain largely unknown. Three specific Aims of the project are proposed in order to advance our understanding of the cellular and molecular mechanisms of IK1 and Kir2 channel function. Aim 1 will study the effects of Kir2 heteromerization using covalently linked Kir2 tetrameric concatemers with fixed subunit stoichiometry. In particular, experiments will test a hypothesis whether individual Kir2 subunits exert a dominant effect on specific properties of the channel. In Aim 2 the membrane distribution of lK1 channels, a critical parameter to their function, will be determined. We will employ genetically engineered mice and novel electrophysiological approaches to test the hypothesis that the properties of lK1 are strongly dependent on membrane-specific expression of distinct Kir2 subunits in the outer sarcolemma, t-tubules or intercalated disks. Aim 3 will study IK1 regulation in order to uncover the mechanisms underlying a novel phenomenon of early AP shortening during acute hypoxia, a field currently dominated by a paradigm of KATP channel activation. Specifically, Aim 3 will test the hypothesis that early AP shortening is mediated by up-regulation of IK1, which precedes the late activation of KATP channels. It becomes increasingly clear that upregulation of IK1 may be highly proarrhythmic and thus understanding the mechanisms of IK1 function is of great importance for the development of novel approaches in the treatment and prevention of cardiac arrhythmias.